Exogenous surfactant administration in mice exposed to MV only did not affect peak inspiratory pressure PIP, lung IL-6 levels and the development of perfusate inflammation compared to no
Trang 1R E S E A R C H A R T I C L E Open Access
The effects of exogenous surfactant administration
on ventilation-induced inflammation in mouse
models of lung injury
Valeria Puntorieri1*, Josh Qua Hiansen1, Lynda A McCaig3, Li-Juan Yao3, Ruud AW Veldhuizen1,2,3
and James F Lewis1,2,3
Abstract
Background: Mechanical ventilation (MV) is an essential supportive therapy for acute lung injury (ALI); however it can also contribute to systemic inflammation Since pulmonary surfactant has anti-inflammatory properties, the aim
of the study was to investigate the effect of exogenous surfactant administration on ventilation-induced systemic inflammation
Methods: Mice were randomized to receive an intra-tracheal instillation of a natural exogenous surfactant preparation (bLES, 50 mg/kg) or no treatment as a control MV was then performed using the isolated and perfused mouse lung (IPML) set up This model allowed for lung perfusion during MV In experiment 1, mice were exposed to mechanical ventilation only (tidal volume =20 mL/kg, 2 hours) In experiment 2, hydrochloric acid or air was instilled intra-tracheally four hours before applying exogenous surfactant and ventilation (tidal volume =5 mL/kg, 2 hours)
Results: For both experiments, exogenous surfactant administration led to increased total and functional surfactant in the treated groups compared to the controls Exogenous surfactant administration in mice exposed to MV only did not affect peak inspiratory pressure (PIP), lung IL-6 levels and the development of perfusate inflammation compared to non-treated controls Acid injured mice exposed to conventional MV showed elevated PIP, lung IL-6 and protein levels and greater perfusate inflammation compared to air instilled controls Instillation of exogenous surfactant did not influence the development of lung injury Moreover, exogenous surfactant was not effective in reducing the concentration of inflammatory cytokines in the perfusate
Conclusions: The data indicates that exogenous surfactant did not mitigate ventilation-induced systemic
inflammation in our models Future studies will focus on altering surfactant composition to improve its
immuno-modulating activity
Keywords: Acute lung injury, Mechanical ventilation, Exogenous surfactant, Systemic inflammation
Background
Pulmonary surfactant is a mixture of phospholipids,
surfactant-associated proteins and neutral lipids which
has an important role in the lung in both host defence
mechanisms such as modulating pulmonary
inflamma-tion and in stabilizing the alveoli by reducing surface
tension [1,2] Both biophysical and immuno-modulatory
properties of endogenous surfactant are essential for
normal lung function Importantly, both properties are se-verely impaired during the course of acute lung injury (ALI) [3,4]
ALI is a life threatening condition characterized by bi-lateral pulmonary infiltrates on chest radiograph, alveo-lar edema and hypoxemia [5] Mortality is approximately 30-40%, with the main cause of death resulting from multiple organ failure (MOF) rather than respiratory failure The former is thought to develop in large part due to the release of inflammatory mediators from the lung into the circulation thereby contributing to excessive
* Correspondence: vpuntori@uwo.ca
1
Department of Physiology & Pharmacology, Western University, London,
Ontario, Canada
Full list of author information is available at the end of the article
© 2013 Puntorieri et al.; licensee BioMed Central Ltd This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
Trang 2systemic inflammation This, in turn, causes MOF and
death [6-8]
The main supportive therapy required to maintain
ad-equate oxygenation for patients with ALI is mechanical
ventilation (MV) Unfortunately, this intervention is also
an important component of the complex
pathophysi-ology of ALI, since it can increase pulmonary
inflamma-tion and contribute to the development of the associated
systemic inflammation leading to MOF [9-13] A
pharma-cological therapy capable of mitigating the specific
inflam-matory effects of MV thereby reducing the contribution of
the lung to the systemic inflammation is needed Based on
the known properties of surfactant within the lung, the
current study investigated on such potential therapy
namely exogenous surfactant administration
Exogenous surfactant has been investigated as a
pos-sible therapy for ALI in many experimental and clinical
studies [14-17] Traditionally surfactant treatment has
been administered to improve the biophysical function
of this material within the lung Although extensive
re-search has shown improvements in physiological and
bio-physical outcomes following surfactant treatment, there
was no effect on mortality [18] Contrasting this
exten-sively investigated approach, only a limited number of
studies have evaluated surfactant with the aim to
down-regulate the systemic inflammation associated with ALI
and MV Previous studies in our laboratory demonstrated
that elevated endogenous surfactant pool sizes prior to
MVattenuated the development of pulmonary and
sys-temic inflammation in animal models where injurious
MV was applied to normal lungs [19] or conventional
ventilation was applied to lungs with a pre-existing
in-jury (lipopolysaccharide-induced ALI) [20] Whether
exogenous surfactant can mirror these observations
obtained with elevated endogenous surfactant is not
known It was therefore hypothesized that
administra-tion of exogenous surfactant prior to MV would
re-duce the systemic inflammation associated with lung
injury
To test this hypothesis, two separate mouse models
were utilized: i) a model of mechanical ventilation in
animals with otherwise normal lungs and ii) a model
of acid-induced lung injury followed by MV For both
experiments, exogenous surfactant was administered
prior toMV, and the ventilation was performed ex vivo
using an isolated and perfused mouse lung (IPML)
setup The inflammatory mediators released by the
lungs into the circulation were collected (via left
ven-tricle) in perfusate and re-circulated (via pulmonary
artery) throughout MV This ex vivo circulatory
sys-tem in the IPML setup allowed us to isolate the
contri-bution of mechanically ventilated lungs to the systemic
system, with perfusate representing a surrogate of
sys-temic inflammation
Methods
Experimental design and ethics statement
A total of 36 male 129X1/SVJ mice (Jackson Laborator-ies, Bar Harbor, Me., USA) were utilized for two separate animal experiments All procedures were approved by the Animal Use Subcommittee at Western University (Permit Number: 2010–272) and, whenever necessary, adequate anesthetic regimen was used to minimize suf-fering For both experiments, mice were allowed to acclimatize for a minimum period of 72 hours in an ani-mal facility, during which time they were allowed free access to water and standard chow
In order to test our hypothesis of an anti-inflammatory role of surfactant toward the effects of MV, administration
of exogenous surfactant was performed in two separate models of lung injury: experiment 1 involved the use of
MV only and experiment 2 involved the use of intra-tracheal (i.t.) instillation of hydrochloric acid (HCl) followed by conventional MV
In experiment 1, mice were anaesthetized and subse-quently randomized to either exogenous surfactant ad-ministration or no treatment After the completion of the i.t surfactant instillation, mice were connected to the IPML setup and exposed immediately following re-perfusion to MV with a tidal volume (Vt) of 20 ml/kg, a positive end expiratory pressure (PEEP) of 3 cmH2O, and a respiratory rate (RR) of 30 breaths/min This re-sulted in the randomization of a total of 12 mice to one
of the two experimental conditions: i) No Treatment group or ii) bLES group
In experiment 2, a total of 24 male 129X1/SVJ mice were anaesthetized and then randomized to receive an intra-tracheal instillation of HCl or air Four hours after the development of acid-induced lung injury, mice were randomized to receive an intra-tracheal exogenous sur-factant administration (or no treatment) before ex vivo,
in situ MV The IPML setup was used to ventilate these animals with the following ventilation parameters: Vt =
5 ml/kg, PEEP = 3 cmH2O, RR = 60 breaths/min This resulted in the following experimental conditions: i) air + no treatment; ii) air + bLES; iii) acid + no treat-ment; iv) acid + bLES
Intra-tracheal hydrochloric acid instillation
Mice were randomized to receive either an intra-tracheal (i.t.) administration of HCl or air as a control, as previ-ously described [9] Briefly, mice were anesthetised with
an intra-peritoneal injection of ketamine (130 mg/kg; Sandoz, Quebec, Que., Canada) and xylazine (6 mg/kg; Bayer, Toronto, Ont., Canada) Once the proper depth
of anesthesia was reached, mice were positioned dor-sally on a vertical stand and their trachea was intubated with a 20-gauge catheter coupled with a fiber-optic stylet (BioLite intubation system for small rodents, BioTex, Inc.,
Trang 3Houston, Tex., USA) Animals randomized to the acid
in-stillation group were given 50μl of 0.05 Ν HCl in a
drop-wise fashion through the endotracheal tube Animals
randomized to the control group were intubated as
de-scribed and allowed to breathe spontaneously through
the tube The total procedure took approximately 5 minutes
Mice were then extubated, positioned on a horizontal
in-clined stand and administered sub-cutaneous injections of
buprenorphine (0.05-0.1 mg/kg) and 1 ml of sterile normal
saline Subsequently, mice were returned to the cage and
allowed to recover for 4 hours with free access to water and
food Mice were carefully monitored during the 4 hours
re-covery period
Intra-tracheal surfactant instillation
Mice were anesthetised with an intra-peritoneal (i.p.)
in-jection of ketamine (130 mg/kg) and xylazine (6 mg/kg)
Animals were then positioned dorsally on a vertical
ro-dent stand and the trachea was intubated trans-orally
with a 20-gauge catheter coupled with a fiber-optic stylet
(BioLite intubation system for small rodents, BioTex,
Inc., Houston, Tex., USA) Mice randomized to the
sur-factant administration group were given 50 mg/kg bLES
(BLES Biochemicals, London, Ont., Canada) in a drop
wise fashion through the endotracheal tube This
nat-ural, bovine lipid extracted surfactant is composed of
ap-proximately 97% phospholipids, 3% neutral lipids, and
about 1% by weight proteins [21] After the surfactant
was spontaneously inhaled by the animals, mice were
extubated and positioned on a horizontal inclined stand
To allow for peripheral surfactant distribution, based on
preliminary experiments, mice were allowed to
spontan-eously breathe for 12–15 minutes before MV Animals
randomized to the no treatment group were intubated
as described and allowed to breathe spontaneously
Isolated and perfused mouse lung setup
Mice were ventilated for a total of 2 hours using the
IPML setup Following exogenous surfactant
administra-tion (or no treatment), the anesthetised mice were sacrificed
with an additional i.p injection of ketamine (200 mg/kg)
and xylazine (10 mg/kg) A tracheostomy tube was then
inserted and secured in the trachea, and the animals were
subsequently connected to the IPML apparatus as described
by Von Bethmann et al [22] Briefly, the heart and lungs
were surgically exposed and the lungs were ventilated with a
volume cycled, positive pressure ventilator (Flexivent, Scireq,
Montreal, Que., Canada) with different ventilation strategies
as described in detail under the experimental design section
Perfusate (RPMI lacking phenol red + 2% w/v low endotoxin
grade Bovine Serum Albumin; Sigma, St Louis, Mo., USA)
was circulated into the pulmonary vasculature through a
catheter inserted in the pulmonary artery and collected by a
second catheter in the left ventricle Once the lungs were
cleared of all the blood, perfusate was delivered in a re-circulating fashion (rate 1 ml/min) during the 2 hours of
MV One milliliter of perfusate was collected at baseline (time 0, immediately after vascular clearing and before perfusate re-circulation) and every 30 minutes of MV thereafter Samples were frozen and stored at −80°C for subsequent measurement of inflammatory media-tors Physiological parameters such as peak inspiratory pressure (PIP) and perfusion pressure were monitored throughout ventilation utilizing Chart v.4.12 software (AD Instruments, Castle Hill, Australia)
Surfactant and total lung lavage protein measurements
Immediately after MV using the IPML setup, lungs were lavaged with 3 × 1 ml aliquots of 0.9% NaCl solution with each aliquot instilled and withdrawn 3 times The total lavage volume was recorded and average recoveries
of lavage fluid were 2.7 mL and 2.8 mL for experiment 1 and experiment 2, respectively Total lavage was then immediately centrifuged at 380 g for 10 min at 4°C to remove the cellular component, and the collected super-natant was termed total surfactant (TS) A 1 ml aliquot
of TS was stored at−80°C for cytokine and protein ana-lysis In order to separate the small aggregate sub-fraction (SA) from the large aggregate (LA) sub-fraction, 1 ml of
TS was centrifuged at 40,000 g for 15 min at 4°C The LA pellet was then re-suspended in 0.3 ml of 0.9% NaCl, while the supernatant represented the SA fraction The leftover volume of TS was used for analysis of total surfactant pool size TS, LA and SA were frozen and stored at−80°C Measurement of the phospholipid content in TS, LA and
SA was performed by phosphorous assay on chloroform-methanol extracted samples, as previously described [23,24] Total protein content in lavage was assessed using a Micro BCA protein assay kit (Pierce, Rockford, Ill., USA) according
to manufacturer’s instructions
Biophysical functional analysis of surfactant
LA sub-fractions from animals within each experimental group were pooled together for functional analysis An aliquot from each pooled sample was utilized to measure the total phospholipid content by phosphorous assay, while the remaining pooled LA was centrifuged at 40,000 g for 15 min at 4°C The supernatant was then discarded and the purified LA pellet re-suspended in a buffer solution (1.5 mM CaCl2, 5 mM TRIS) to a final phospholipid concentration of 5 mg/ml The surface ac-tivity of the LA samples was assessed using a computer-controlled captive bubble surfactometer (CBS, 3 runs for each pooled sample) as previously described [25,26]
Measurement of inflammatory mediators
Interleukin-6 (IL-6) levels were measured in aliquots
of lung lavage and in perfusate aliquots obtained at
Trang 4different time points using an enzyme-linked
immuno-sorbent assay (ELISA) kit following manufacturer’s
in-structions (BD Biosciences, San Diego, CA., USA) A
broader array of inflammatory mediators was
mea-sured in perfusate samples collected at the end of MV
using a Milliplex Map mouse cytokine/chemokine panel
(MPXMCYTO-70 K-12; Millipore Corporation, Billerica,
MA, USA) for the following 12 analytes: eotaxin,
granulo-cyte colony-stimulating factor (G-CSF), granulogranulo-cyte-
granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1β,
IL-6, IL-13, interferon-γ-induced protein 10 (IP-10),
keratinocyte chemoattractant (KC),
lipopolysaccharide-induced CXC chemochine (LIX), monocyte chemotactic
protein-1 (MCP-1), macrophage inflammatory protein 2
(MIP-2) and tumor necrosis factor-alpha (TNF-α)
Sam-ples were analyzed utilizing the Luminex® xMAP®
detec-tion system on the Luminex100 (Linco Research, St
Charles, Mo., USA) according to the manufacturer’s
in-structions Perfusate samples collected at the end of MV
in experiment 2 were further analyzed for eicosanoids
levels (8-isoprostane, prostaglandin E2, leukotriene B4,
thromboxane B2) using colorimetric competitive
en-zyme immunoassay (EIA) kits (Cayman Chemical
Com-pany, Ann Arbor, MI, USA) according to manufacturer’s
instructions
Statistical analysis
All data are expressed as mean ± standard error of the
mean (SEM) Statistical analyses were performed using
the GraphPad Prism statistical software (GraphPad
Soft-ware, Inc., La Jolla, CA., USA) Data were analysed with
a t-test or one way ANOVA with a Tukey’s post hoc test
when appropriate (experiment 1) For experiment 2, a
two-way ANOVA (variables: presence of primary insult
and treatment effects) followed by a one-way ANOVA
with a Tukey’s post hoc test was used to analyse the
data A repeated measures two-way ANOVA was
per-formed when appropriate with a Bonferroni post hoc
test P < 0.05 was considered statistically significant
Results
Experiment 1
In experiment 1 the effects of exogenous surfactant
ad-ministration on lung and systemic inflammation during
MV of otherwise normal lungs were determined Peak
inspiratory pressure (PIP) was recorded throughout MV
PIP ranged between 20.62 ± 1.6 cmH2O and 22.6 ± 2.7
cmH2O for the No Treatment group (time 0 and time
120 min, respectively) and varied between 22.6 ± 2.7
cmH2O and 26.3 ± 2.7 cmH2O for the bLES group (time
0 and time 120 min, respectively) Exogenous surfactant
administration did not reduce PIP values in the
surfac-tant treated group compared to No Treatment
Perfu-sion pressure was also monitored throughout MV and
maintained between 4 and 6 mmHg for both groups (data not shown)
Lavage analysis
Results reflecting local inflammation, as assessed by pul-monary permeability changes and inflammatory markers are shown in Table 1 The total protein content and IL-6 levels in lung lavage collected at the end of MV were not affected by surfactant treatment, with no statistically significant differences noted in these values between bLES treated and non-treated groups Recoveries of lung lavage fluid were not statistically significant between groups (data not shown)
Surfactant pool sizes of TS, LA and SA sub-fractions isolated from lung lavage for the two groups are shown
in Figure 1A As expected, TS pools were significantly higher in the bLES treated group compared to No Treat-ment mice Similarly to TS values, LA and SA pools were significantly higher in the bLES group compared to the No Treatment group (Figure 1A)
The functional activity of the LA samples measured during four different dynamic compression-expansion cycles is shown in Figure 1B for each experimental group No significant differences in surface tension were found between bLES treated and No Treatment mice for any of the cycles Within each group, the minimum achievable surface tension was significantly higher dur-ing cycle 10 compared with cycles 1 and 2 (Figure 1B)
Perfusate analysis
The concentration of IL-6 was measured in perfusate samples in order to assess the effects of exogenous sur-factant on the development of systemic inflammation (Figure 2) IL-6 levels were not detectable within the first
30 minutes of MV (time 0 and 30 min; data not shown)
A gradual increase in perfusate IL-6 was measured at
60 and 90 minutes in both groups; however, there was
no statistically significant difference in this cytokine level between bLES treated and No Treatment mice at any time point throughout MV
Perfusate concentrations of 11 cytokines/chemokines measured at the end of MV by multiplex assay are shown in Table 2 Perfusate IL-13 levels were not detect-able (data not shown) There was no statistically signifi-cant effect of exogenous surfactant administration on
Table 1Experiment 1: Total protein levels and IL-6 concentrations in lung lavage at the end of MV
Mechanical ventilation
Total lavage protein (mg/kg body weight)
Values are expressed as mean ± SEM; n = 6 per group.
Trang 5cytokines/chemokines concentrations in perfusate, with
no differences between No treatment and bLES groups
Experiment 2
In experiment 2, the effect of exogenous surfactant on
sys-temic inflammation during MV was assessed in the presence
of a pre-existing acid-induced lung injury/inflammation
Physiological parameters such as peak inspiratory pressure
and perfusion pressure were monitored throughout
ventila-tion as in experiment 1, and PIP values are shown in
Figure 3 Although all experimental groups were
ex-posed to the same ventilation strategy, the peak
in-spiratory pressure was significantly higher in Acid injured
mice compared to the respective Air groups (Acid No Treatment vs Air No Treatment; Acid bLES vs Air bLES) Exogenous surfactant administration led to a significant increase in PIP values during the first hour of MV (10 to
75 min) in the Air bLES group compared to Air No Treat-ment group and, importantly, did not reduce PIP values in
Figure 1 Experiment 1: Surfactant recovery in lung lavage and surface activity of LA A: surfactant pool size of TS, LA and SA sub-fractions measured by phosphorous assay Data are expressed as amount of phospholipids/kg body weight Within each sub-fraction, *p < 0.05 vs the No Treatment condition B: minimum surface tension of pooled LA samples during different dynamic compression-expansion cycles #p < 0.05 versus cycle 1 and 2 within each experimental condition Values are expressed as mean ± SEM.; n = 6 per group.
Figure 2 Experiment 1: IL-6 levels measured in lung perfusate
at 60, 90 and 120 min Values are expressed as mean ± SEM.; n = 6
per group.
Table 2Experiment 1: Cytokine and chemokine analysis
in lung perfusate at the end of MV
Mechanical ventilation
G-CSF = granulocyte colony-stimulating factor, GM-CSF = granulocyte-macrophage CSF, IL-6 = interleukin-6, IP-10 = interferon-γ-induced protein 10, KC = keratinocyte chemoattractant, LIX = lipopolysaccharide-induced CXC chemokine, MCP-1 = monocyte chemotactic protein-1, MIP-2 = macrophage inflammatory protein 2 and TNF-α = tumor necrosis factor-alpha.
Trang 6the Acid bLES group compared to Acid No Treatment
group at any time point Perfusion pressure was
moni-tored during MV and maintained between 5 and 7 mmHg
for all groups (data not shown)
Lavage analysis
Lung permeability, as reflected by total protein content
in lung lavage (Table 3), was significantly higher in the
acid injured animals versus the air control groups, whether
they were given surfactant or not (Acid No Treatment vs
Air No Treatment; Acid bLES vs Air bLES) No significant
difference was noted between Air bLES versus Air No
Treatment and Acid bLES versus Acid No Treatment
Similar results were observed for IL-6 concentration in
lung lavage (Table 3) Acid–instilled animals showed
greater IL-6 levels in lavage compared to the respective
air-instilled controls Exogenous surfactant did not affect
lavage IL-6 levels in both air groups (Air bLES vs Air No
Treatment); however, there was a significantly higher
cyto-kine concentration in the lavage of Acid bLES mice
com-pared to the Acid No Treatment group Recoveries of
lung lavage fluid were not statistically significant between
groups (data not shown)
Surfactant sub-fractions and the surface activity of iso-lated LA are shown in Figures 4A and B respectively Acid instillation did not change TS, LA and SA pool sizes compared to their respective Air control groups (Figure 4A) This was similar for both not treated and surfactant treated groups As expected and observed in experiment 1, total surfactant and LA values were sig-nificantly higher in surfactant treated groups than non-surfactant treated controls (Air bLES vs Air No Treatment; Acid bLES vs Acid No Treatment) There was no difference in SA values among the various ex-perimental groups
There were no statistically significant differences noted between any of the experimental groups in the biophys-ical activity of the LA samples (Figure 4B) Within some
of the groups, however, significant differences in surface tension were measured between the different dynamic cycles In particular, surface tension was significantly higher during compression-expansion of cycles 5 and 10 when compared to cycle 1 within the acid instilled groups (in both Acid No Treatment and Acid bLES) LA from the Air No Treatment and Air bLES groups main-tained low surface tension values throughout the 10 dy-namic compression-expansion cycles
Perfusate analysis
To test the hypothesis of a role for exogenous surfactant
in down-modulating systemic inflammation in ALI, se-quential lung perfusate samples, as a surrogate for systemic inflammation, were analyzed for IL-6 concentrations As shown in Figure 5, there were significantly higher levels of IL-6 in the perfusate of acid-instilled mice compared to the respective air-instilled controls at every time point (0, 30,
60, 90, 120 min; Acid No Treatment vs Air No Treatment; Acid bLES vs Air bLES) Perfusate IL-6 levels were not significantly affected by exogenous surfactant adminis-tration, with no differences between Air bLES and Air
No Treatment and no change between Acid bLES and Acid No treatment
Lung perfusate samples collected at 120 min were fur-ther analyzed for a wider array of cytokines/chemokines Among the 12 mediators measured (Table 4), IL-13 levels were not detectable (data not shown), while there were significantly greater levels of eotaxin, IL-6, KC, MIP-2 in acid-instilled animals compared to the respect-ive air instilled control
Figure 3 Experiment 2: Peak Inspiratory Pressure (PIP) was
measured over the course of MV Values are expressed as
mean ± SEM +p > 0.05 versus Air No Treatment at the specific
time point indicated, *p < 0.05 versus the respective Air control at
each time point; n = 6 per group.
Table 3Experiment 2: Total protein levels and IL-6 concentrations were measured in lung lavage at the end of MV
Total lavage protein
(mg/kg body weight)
Trang 7Overall, exogenous surfactant administration did not
affect eotaxin, GM-CSF, IL-6, IL-1β, KC, TNF-α and
IP-10 levels, with no statistical difference between the
bLES and No Treatment group in both Air and Acid
instilled mice
A statistically significant increase of MIP-2 levels in
the perfusate of Acid bLES mice was determined
com-pared to Acid No Treatment, as well as significantly
higher perfusate levels of G-CSF, LIX and MCP-1 in acid
injured mice treated with surfactant compared to the
Air bLES
Finally, in order to further characterize the effect of exogenous surfactant administration on lung-derived mediators in perfusate, eicosanoids levels were also mea-sured at the 120 min time point (Table 5) Although in-creased levels of thromboxane B2 and prostaglandin E2
were recorded in the perfusate of acid-instilled animals compared to their respective Air controls, these changes did not reach statistical significance Perfusate concentra-tions of 8-isoprostane were significantly higher in the acid injured groups compared to air controls Surfactant treat-ment did not affect thromboxane B2 and 8-isoprostane concentrations Prostaglandin E2 levels were significantly elevated only in the perfusate of Acid bLES mice com-pared to Air bLES controls Leukotriene B4levels were in-creased in the perfusate of Acid bLES mice but this difference failed to be statistically significant
Discussion
The overall objective of this study was to evaluate the anti-inflammatory effects of exogenous surfactant when administered prior to mechanical ventilation, either in the absence (experiment 1) or in the presence (experi-ment 2) of an initiating pulmonary insult For both lung injury models, the IPML setup was utilized to specific-ally evaluate the contribution of ventilation to the devel-opment of systemic inflammation MV of normal lungs resulted in the release of IL-6 (locally) into the airspace and several mediators (systemically) in the perfusate Surfactant administration, however, was not effective in reducing the systemic inflammation associated with MV
Figure 4 Experiment 2: Surfactant recovery in lung lavage and surface activity of crude LA A: surfactant pool size of TS, LA and SA sub-fractions measured by phosphorous assay Data are expressed as amount of phospholipids/kg body weight Within each sub-fraction,*p < 0.05 versus the respective No Treatment condition B: surface tension of pooled LA samples during different dynamic compression-expansion cycles §p < 0.05 versus cycle 1 within each experimental condition Values are expressed as mean ± SEM; n = 6 per group.
Figure 5 Experiment 2: IL-6 levels measured in lung perfusate at
0, 30, 60, 90, 120 min Data are expressed as mean ± SEM *p < 0.05
versus respective Air control at each time point; n = 6 per group.
Trang 8Conventional ventilation of HCl instilled mice led to
higher levels of both IL-6 and total protein in lavage,
and significantly higher levels of pro-inflammatory
medi-ators in perfusate without any effect of bLES instillation
Based on these results, it was concluded that
administra-tion of exogenous surfactant prior to MV does not
re-duce the systemic inflammation associated with lung
injury in these models
An important feature of the current study was to
examine the effects of surfactant therapy in two different
models Analysis of the data showed important
differ-ences between the models, such as the degree of lung
edema Mechanical stretch of uninjured lungs did not
affect lung permeability, whereas acid injured mice had
increased total lavage proteins after two hours of MV
Another aspect that distinguishes the two models is
rep-resented by different levels of pulmonary and perfusate
inflammation, which becomes particularly evident when
comparing cytokine levels measured in the perfusate of
the MV only, No Treatment group to the cytokine levels
of the Acid No Treatment group For example, MV of
normal lungs caused a moderate increase in circulating Eotaxin, IL-6, KC and MIP-2, while acid instilled animals subjected to conventional MV had perfusate concentra-tions of these mediators that were at least two times greater Given the greater inflammation characterizing the acid-injury model and the important role of lipid mediators in the development and progression of lung injury [27-32], eicosanoids levels were analyzed only on samples from experiment 2 Unambiguous conclusions about the effects of exogenous surfactant on systemic in-flammation were therefore inferred from two experimen-tal models with very different characteristics This allowed
us to rule out possible causes for the lack of efficacy of our treatment (such as presence/lack of pre-existing in-jury, specific effects of ventilation), and strengthened the understanding of the biological response
Exogenous surfactant administration has been exten-sively investigated as a potential adjunctive therapy in acute lung injury [33-36] The traditional approach with surfactant treatment has been to evaluate its efficacy in terms of physiological and biophysical improvements
Table 4Experiment 2: Cytokine and chemokine levels measured in lung perfusate at the end of MV
G-CSF = granulocyte colony-stimulating factor, GM-CSF = granulocyte-macrophage CSF, IL-6 = interleukin-6, IP-10 = interferon- γ-induced protein 10, KC = keratinocyte chemoattractant, LIX = lipopolysaccharide-induced CXC chemokine, MCP-1 = monocyte chemotactic protein-1, MIP-2 = macrophage inflammatory protein 2 and TNF-α = tumor necrosis factor-alpha.
Data are expressed as mean ± SEM; n = 6 per group *p > 0.05 versus respective Air control, #p < 0.05 versus Acid No Treatment.
Table 5Experiment 2: Concentrations of prostaglandin E2, leukotriene B4, thromboxane B2and 8-isoprostane measured
in lung perfusate samples collected at the end of MV
Trang 9Many experimental studies have in fact demonstrated
that exogenous surfactant instilled after the onset of
ventilation improved oxygenation, lung volume and
compliance; moreover, it improved the surface tension
reducing properties of the surfactant recovered from
lung lavage subsequent to administration [15,37,38]
Nevertheless, despite this encouraging experimental
evi-dence, clinical trials showed no improvement in
mortal-ity in surfactant treated patients even in the presence of
an initial improvement in oxygenation [16,18] It is
pos-sible that surfactant treatment in the previous studies
was administered too late into ALI development;
there-fore earlier surfactant administration prior to or at the
onset of MV could be more effective at mitigating
dis-ease progression Since mortality can be improved by
ameliorating ventilation – induced systemic
inflamma-tion [39], it was our interest to investigate whether
ex-ogenous surfactant could mitigate the effects of MV
thereby down-modulating inflammation
To our knowledge, the effect of surfactant on
ventilation-induced release of inflammatory mediators in perfusate of
an IPML model has been specifically addressed in two
pre-vious studies Stamme and colleagues [40] showed elevated
TNFα and IL-6 concentrations in the perfusate of
surfac-tant treated animals compared to controls, in their mouse
model of high pressure ventilation In contrast, our group
has shown a reduced level of inflammatory cytokines in
perfusate due to elevated endogenous surfactant in an LPS
model of injury [20] Together with the current study in
which surfactant did not impact inflammation in two
models of injury, these data illustrate the complexity of
sur-factant treatment in which specific details of the
experi-mental model may impact outcome Furthermore, such
details are obviously important to understand in the
con-text of a potential clinical utilization of surfactant treatment
to down-regulate systemic inflammation as well as to
understand the mechanisms by which surfactant may affect
inflammation
Despite the lack of effect of surfactant treatment in
our study, we speculate that mitigation of MV induced
inflammation is still the best approach for an early
inter-vention Our data support earlier studies which showed
that cytokines can be detected in perfusate rapidly after
the onset of ventilation [22,41] This loss of alveolar and
systemic cytokine compartmentalization can lead to
per-ipheral organ dysfunction, a condition of difficult clinical
management Therefore, targeting the lung with
anti-inflammatory agents prior to MV may be a successful
treatment option leading to improved outcomes In this
respect, surfactant could be utilized as a carrier for
deliv-ering lung specific anti-inflammatory agents prior to
MV in future studies
Along with the strengths of the present study, some
limitations need to be addressed Due to the lack of
blood perfusion in the IPML setup, the lungs were not exposed during ex vivo MV to circulating soluble factors and immune cells which could have affected the pro-gression of the injury Moreover, ex vivo ventilation of perfused lungs did not favor the use of severe lung injury models, due to potential technical failure of the prepar-ation Consequently, the injury from ventilation was mild to moderate, thereby explaining the lack of change
in surface tension or surfactant pool sizes It is believed, however, that these limitations of the IPML setup were counter balanced by the advantage of specifically isolat-ing lung-derived mediators released into the circulation, without the confounding contribution of systemic fac-tors to the development of inflammation Intra-tracheal instillation was also used for administering surfactant, ensuring the presence of large amounts of active mater-ial in the airspace before ventilation, as shown by higher levels of TS and LA in the lung lavage of treated ani-mals It should be acknowledged, however, that some in-adequate distribution of surfactant might have occurred following instillation Obstruction of smaller airways, with consequent heterogeneous lung inflation and re-gional over-distension might have been responsible for the increase in PIP (experiment 2, Air treated groups), and for the non-significant trend towards higher lavage IL-6 levels in the surfactant treated groups Nevertheless, the instilled surfactant retained excellent biophysical prop-erties as shown by the very low minimum surface tension achieved during dynamic compression-expansion of the crude LA Overall, we believe that instillation did not ac-count for the lack of efficacy of our treatment
Conclusions
In conclusion, this study expands the knowledge about exogenous surfactant treatment It specifically focuses
on the anti-inflammatory effects of a lung targeted therapy administered prior to MV on the development of systemic inflammation using two different mouse models Although our data suggest a lack of efficacy for exogenous surfactant
in down-modulating inflammation, future studies might focus on surfactant as a carrier for anti-inflammatory drugs or antibiotics in order to better interfere with ALI progression
Abbreviations
ALI: Acute lung injury; MV: Mechanical ventilation; MOF: Multi-organ failure; bLES: Bovine lipid extract surfactant; IPML: Isolated and perfused mouse lung; PIP: Peak inspiratory pressure; Vt: Tidal volume; PEEP: Positive end expiratory pressure; RR: Respiratory rate.
Competing interests The authors declare that they have no competing interests.
Authors ’ contributions
VP – Experimental procedures and design, data analysis, manuscript writing JQH - Experimental procedures, data analysis, manuscript review LAM - Experimental procedures LJY - Experimental procedures RAWV - Experimental
Trang 10design, data analysis, manuscript review JFL - Experimental design, data analysis,
manuscript review All authors read and approved the final manuscript.
Acknowledgments
The authors thank the rest of the Lung Lab for helpful discussion We are
grateful to bLES Biochemical Inc London, Ontario for generously providing
bLES Financial support was provided by the Canadian Institutes of Health
Research.
Author details
1
Department of Physiology & Pharmacology, Western University, London,
Ontario, Canada 2 Department of Medicine, Western University, London, ON,
Canada.3Lawson Health Research Institute, London, ON, Canada.
Received: 29 January 2013 Accepted: 14 November 2013
Published: 20 November 2013
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